Modulation of Double-Negative T Cells by Huang-Lian-Jie-Du Decoction Attenuates Neuroinflammation in Ischemic Stroke:Insights from Single-Cell Transcriptomics

Wang Kai Zijin Sun Qi Shao Zhaoyi Wang Haojia Zhang Yuanhua Li Jingmei Ming Wenjing Zhang Tieshan Wang Yan Zhao ^* Qingguo Wang ^* Fafeng Cheng ^*

• Beijing University of Chinese Medicine, Beijing, China

Ischemic stroke(IS) is a major global health issue characterized by high rates of morbidity and mortality, largely driven by inflammatory responses. Double-negative T cells (DNTs), a unique subset of T cells lacking CD4 and CD8 markers, have shown potential dual roles in IS pathogenesis, though their precise functions remain unclear. In this study, we investigated the effects of DNTs during the acute phase of IS and examined how Huang-Lian-Jie-Du Decoction (HLJD), a traditional Chinese medicine formula, influences these cells. Using single-cell transcriptomics, we identified two distinct DNT subtypes: an activated, cytotoxic phenotype (Kill+) and a resting, immunosuppressive phenotype (Kill-). Our results demonstrate that HLJD treatment can modulate the balance of these DNT subtypes, reducing the proportion of cytotoxic DNTs and increasing immunosuppressive DNTs. This shift was associated with decreased immune infiltration and inflammation in brain tissue, potentially leading to reduced neuronal damage. These findings suggest that HLJD exerts neuroprotective effects in IS by modulating the activity and distribution of DNT cells, offering new insights into the therapeutic potential of traditional medicine for IS. Further research is warranted to clarify the mechanisms underlying DNT-mediated immune responses in IS and to explore the broader applications of HLJD in neuroinflammatory diseases.